Figure 4.

Characterization of MuRF1^B2|CC|COS. (a) Electron micrograph of negatively stained MuRF1^B2|CC|COS samples corresponding to the outermost tail fractions of size-exclusion chromatograms containing dimeric assemblies (electronic supplementary material, figure S3). (b) Gallery of three major class averages obtained by using the processing software EMAN1. (c) Complexation of MuRF1^H1 and MuRF1^COS samples monitored by size-exclusion chromatography. The complex was formed by mixing the samples in a molar ratio of 1 : 2.5 in 20 mM Tris–HCl pH 7.5, 200 mM NaCl, 1 mM DTT followed by incubation for 1 h at 4°C. The mixture was run on a Superdex 200 HiLoad 26/60 column. Chromatogram and associated SDS-PAGE are shown. MW marker is SeeBlue Plus2 Pre-stained standard (Invitrogen) (samples are proximal to the 6 kDa band). (d) Proposed quaternary structure of MuRF1 compiling known and predicted structural information on MuRF1. The structure of the B2-box dimer is that previously elucidated by X-ray crystallography (PDB 3DDT) [29]; the model for the HD and its dimeric assembly is as deducted in the current study.